Báo cáo y học: " Specific TATAA and bZIP requirements suggest that HTLV-I Tax has transcriptional activity subsequent to the assembly of an initiation complex" pps

Conclusions: Optimal activation of the HTLV-I LTR by Tax specifically requires the core HTLV-I TATAA promoter, CREB and the 21-bp repeats.. Each reporter contains two copies of enhancer

Bio Med Central

Retrovirology

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Research

Specific TATAA and bZIP requirements suggest that HTLV-I Tax has transcriptional activity subsequent to the assembly of an

initiation complex

Address: 1 Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong, China, 2 Department of Pathology, The University

of Hong Kong, Pokfulam, Hong Kong, China, 3 National Key Laboratory for Molecular Virology, Institute of Virology, 100 Yingxin Street, Beijing

100052, China and 4 Laboratory of Molecular Microbiology, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-0460, USA Email: Yick-Pang Ching - ypching@hkucc.hku.hk; Abel CS Chun - cschun@hkusua.hku.hk; King-Tung Chin - tonychin@hkusua.hku.hk;

Zhi-Qing Zhang - zhangzq@public3.bta.net.cn; Kuan-Teh Jeang - kj7e@nih.gov; Dong-Yan Jin* - dyjin@hkucc.hku.hk

* Corresponding author

Abstract

Background: Human T-cell leukemia virus type I (HTLV-I) Tax protein is a transcriptional

regulator of viral and cellular genes In this study we have examined in detail the determinants for

Tax-mediated transcriptional activation

Results: Whereas previously the LTR enhancer elements were thought to be the sole Tax-targets,

herein, we find that the core HTLV-I TATAA motif also provides specific responsiveness not seen

with either the SV40 or the E1b TATAA boxes When enhancer elements which can mediate

Tax-responsiveness were compared, the authentic HTLV-I 21-bp repeats were found to be the most

effective Related bZIP factors such as CREB, ATF4, c-Jun and LZIP are often thought to recognize

the 21-bp repeats equivalently However, amongst bZIP factors, we found that CREB, by far, is

preferred by Tax for activation When LTR transcription was reconstituted by substituting either

κB or serum response elements in place of the 21-bp repeats, Tax activated these surrogate motifs

using surfaces which are different from that utilized for CREB interaction Finally, we employed

artificial recruitment of TATA-binding protein to the HTLV-I promoter in "bypass" experiments to

show for the first time that Tax has transcriptional activity subsequent to the assembly of an

initiation complex at the promoter

Conclusions: Optimal activation of the HTLV-I LTR by Tax specifically requires the core HTLV-I

TATAA promoter, CREB and the 21-bp repeats In addition, we also provide the first evidence for

transcriptional activity of Tax after the recruitment of TATA-binding protein to the promoter

Background

In eukaryotes, transcription by RNA polymerase II

requires the orderly recruitment of basal transcription

fac-tors and activafac-tors to the core promoter and enhancers,

respectively [1,2] The core promoter contains the tran-scription initiation site, and it provides the docking sites for the basal transcription factors that nucleate the assem-bly of a functional preinitiation complex (PIC) The TATA

Published: 30 July 2004

Retrovirology 2004, 1:18 doi:10.1186/1742-4690-1-18

Received: 27 May 2004 Accepted: 30 July 2004 This article is available from: http://www.retrovirology.com/content/1/1/18

© 2004 Ching et al; licensee BioMed Central Ltd This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

box is one of four major core promoter elements, and it is

specifically recognized by the TATA-binding protein

(TBP), a subunit of the basal transcription factor TFIID

which also contains at least 14 TBP-associated factors

(TAFs) On the other hand, enhancers are bound by

sequence-specific transcriptional activators that are

thought to promote PIC assembly through interactions

with components of the basal transcription machinery

Human T-cell leukemia virus type I (HTLV-I) Tax protein

is a unique transcriptional regulator [3] Tax can modulate

the HTLV-I long terminal repeats (LTR), heterologous

viral promoters, and a variety of cellular genes In most

context, Tax acts as a potent transcriptional activator

through Tax-responsive DNA elements that are

recog-nized by cellular transcription factors CREB, NFκB and

serum response factor (SRF) [4-6] For activation of the

HTLV-I LTR, Tax targets three imperfectly conserved 21-bp

direct repeats flanked by GC-rich sequences In this

sce-nario, Tax forms a ternary complex with CREB and the

21-bp repeat through physical interaction with CREB and

direct contact with the flanking GC-rich sequences [7-9]

Tax-induced activation of other promoters is thought to

be mediated through protein-protein interactions Thus,

Tax is a pleiotropic transcriptional activator that targets

multiple enhancer elements through multiple cellular

transcription factors

To date, the molecular mechanisms for Tax

trans-activa-tion have been well studied Due to its pleiotropic

activi-ties, there are likely nuances to Tax's activity which remain

unrevealed Currently, we understand Tax to harbor a

minimal activation domain [10], to interact with basal

transcription factors such as TBP [11], to form a

homo-dimer [12-14], and to stimulate the homo-dimerization of

cellu-lar regulatory factors such as CREB [15,16] and IKK-γ [17]

Moreover, we also know that Tax can directly engage

tran-scriptional coactivators such as CREB-binding protein,

p300 and P/CAF [18-20] However, it remains unclear

what is Tax's optimal preference for an enhancer – TATAA

configuration It has also been unaddressed whether Tax

has a transcriptional activity after the formation of an

ini-tiation complex at the TATAA-box

In mammalian cells, the artificial recruitment of TBP

suf-ficiently activates transcription from some promoters

[21-24] It is understood that the structure of core promoter is

one important determinant for this activation [23] On

the other hand, DNA-tethered TBP can also work

synergis-tically with selective natural activators such as human

immunodeficiency virus type 1 (HIV-1) Tat protein

[21-23] and cytomegalovirus IE2 protein [25] In this regard,

it is not known whether TBP recruitment suffices for

acti-vation of HTLV-I minimal promoter Nor is it clear

whether Tax can cooperate with promoter-tethered TBP

Here, we have constructed a series of chimeric enhancer-TATAA reporters to analyze the functional roles of these transcription elements in Tax-mediated activation We observed that Tax activates the HTLV-I 21-bp repeats more potently than other enhancer elements Analysis of ten mutants of Tax revealed that Tax utilizes different domains to target different cellular factors We also found that multiple bZIP transcription factors including the newly-identified LZIP are involved in Tax activation of HTLV-I LTR Finally, two other salient findings are that optimal Tax-responsiveness is specified by the HTLV-I-specific TATAA element, and that Tax synergizes with arti-ficially recruited, DNA-tethered, TBP in a phase of tran-scription after the assembly of an initiation complex at the promoter

Results

Specific preference by Tax for only one enhancer element

Tax can activate transcription through 21-bp repeats, CRE,

κB site or SRE [4-9] However, a direct head-to-head com-parison between the relative preferences of Tax for each of these elements is complicated by the context of additional DNA elements in the various promoters tested to date (i.e the HTLV-I LTR versus the HIV-1 LTR versus the inter-leukin-2 promoter) To directly compare enhancer motifs, they should be placed in identical TATAA-context and tested in identical experimental settings Towards this end, we constructed a series of six reporters to dissect the ordered preference of Tax for various enhancers

Each reporter contains two copies of enhancer motifs

(21-bp repeats, CRE, AP1, Sp1, κB or SRE) and a minimal HTLV-I TATAA promoter (Fig 1A) Because all reporters have the same HTLV-1 minimal promoter and are other-wise devoid of any known enhancer elements, side-by-side comparisons would reflect directly the contribution

of the variously added cis-enhancer We observed that the κB- and CRE- motifs had the highest basal activities in HeLa cells in the absence of Tax (Fig 1B, lanes 3, 4, 9 and 10; and Fig 1C, columns 3 and 6 compared to column 1)

Of significant interest, in stark contrast to the cellular CRE elements, the reiterated HTLV-I 21-bp repeats (normally considered as viral CRE elements) and the SRE exerted lit-tle or no basal activity (Fig 1B, lanes 1, 2, 11 and 12; and Fig 1C, lanes 2 and 7 compared to lane 1) The AP1 and Sp1 sites were moderately active (Fig 1B, lanes 5–8 and Fig 1C, lanes 4 and 5) Hence for basal expression in the context of the HTLV-I TATAA promoter, κB, CRE > AP1, Sp1 >> 21 bp, SRE

When the reporters were tested in the presence of Tax, a different pattern emerged Transcription from the 21-bp repeats was stimulated approximately 70-fold (Fig 1D, lane 2 compared to lane 1) while that from the Sp1 site, not prototypically known to be responsive to Tax, was not

Retrovirology 2004, 1:18 http://www.retrovirology.com/content/1/1/18

Relative responsiveness of enhancers to Tax in HeLa cells

Figure 1

Relative responsiveness of enhancers to Tax in HeLa cells (A) CAT reporter plasmid Each plasmid contains two copies of

enhancer elements (21-bp repeats, CRE, AP1, Sp1, κB and SRE) and one copy of HTLV-I minimal promoter (HTLV TATAA)

The enhancer (Enh.) sequences are shown in green (B) A representative example of CAT assay Increasing amounts (5 to 10

µg) of p21-HTLV-CAT (lanes 1 and 2), pCRE-HTLV-CAT (lanes 3 and 4), pAP1-HTLV-CAT (lanes 5 and 6), pSP1-HTLV-CAT (lanes 7 and 8), pKB-HTLV-CAT (lanes 9 and 10) and pSRE-HTLV-CAT (lanes 11 and 12) were transfected into HeLa cells

CAT assays were performed 48 h after transfection AcCM: acetyl chloramphenicol CM: chloramphenicol (C) Basal

transcrip-tional activities of enhancer elements Five microgram of plasmids containing the HTLV TATAA alone (pHTLV-CAT; column 1)

or the indicated enhancer elements (columns 2 to 7) were transfected into HeLa cells and the relative CAT activities were

compared CAT activity from pKB-HTLV-CAT-transfected HeLa cells was taken as 100% (lane 6) (D) Tax-dependent

tran-scriptional activities of enhancer elements The same plasmids as in C plus 1 µg of Tax-expressing plasmid pIEX were co-trans-fected into HeLa cells and the CAT assays were performed Fold activation in the presence of Tax versus in the absence of Tax was calculated and compared All CAT results are representative of three independent experiments

activated significantly over the activity of the HTLV-I

min-imal promoter (Fig 1D, lane 5 compared to lane 1) All

other responses to Tax were markedly weaker than that

seen from the 21-bp repeats Hence, for all practical

pur-poses, only a duplicated 21-bp repeat in the context of

iso-lated placement upstream of an authentic HTLV-I

minimal TATAA box could be regarded as significantly

Tax-responsive in HeLa cells

We repeated the experiments in Jurkat T lymphocytes and

obtained similar results (Fig 2) Thus, while the κB and

CRE enhancers displayed the highest activities in the

absence of Tax (Fig 2B, lanes 3 and 6 compared to lanes

5, 4, 2, 1, and 7), only the 21-bp repeats were highly

responsive to Tax (Fig 2A, lanes 1 and 2; Fig 2C, lane 2)

Our results from HeLa and Jurkat cells consistently

sup-port the preferential activation of the 21-bp repeats by

Tax

Multiple activation surfaces are configured in Tax

In Fig 1D, the 21-bp repeats were activated by Tax >75

fold, while κB and SRE motifs were activated five and

three fold, respectively The low activation of the latter

motifs, although comparatively less significant than that

from the 21 bp elements, was real and reproducible To

further understand how Tax works, we wondered whether

the different magnitudes of activation were due to

quanti-tative or qualiquanti-tative differences in protein-protein

interac-tion To address this question, we examined the separate

responses of the three motifs to a battery of Tax mutants

Previously we had characterized 47 mutations in Tax that

affect transcriptional activity [26] Here we selected 10 of

these Tax mutants to shed light on the discrete surfaces

used by Tax to mediate effects on 21-bp repeats, κB and

SRE All mutants were expressed to comparable levels in

HeLa cells (data not shown) Their relative activities on

21-bp repeats, κB and SRE were assessed (Fig 3)

Based on percentage of activation relative to wild type Tax,

we saw three patterns of mutant activity for 21 bp, κB and

SRE (Fig 3) Hence, the activation domain mutant Tax

L320G [10] and the zinc finger mutant Tax H52Q [26]

were defective in activating either 21-bp repeats or SRE,

but were fully competent for κB (Fig 3, lanes 4 and 10)

By contrast, the N-terminal mutant Tax ∆3–6 and the

point mutant Tax S258A activated 21-bp repeats and SRE

well, but did not activate κB (Fig 3, lanes 2 and 7)

Addi-tionally, mutants Tax ∆94–114, Tax S150A and Tax ∆337–

353 were active on all three motifs (Fig 3, lanes 5, 6 and

11), while neither Tax ∆2–58, Tax ∆ 284–353 nor Tax

L296G (Fig 3, lanes 3, 8 and 9) activated any of the

motifs These non-identical patterns suggest that Tax may

use different contact surfaces to target factors docked at

the 21-bp repeats, κB or SRE We note some similarity in

the Tax mutant activity profiles for the 21-bp repeats and SRE suggesting that overlapping surfaces may be utilized

Amongst bZIP factors, CREB is specifically preferred by Tax

Tax activates the HTLV-I LTR through the viral 21-bp repeats [7-9] When compared to κB and SRE, the activa-tion of 21-bp repeats by Tax is particularly effective (Fig 1 and Fig 2) and, based on mutant profiles (Fig 3A), relies upon unique structural surfaces Previously, it has been proposed that bZIP cellular transcription factors including CREB [9,27,28], ATF4 [29,30] and c-Jun [31] play roles in Tax activation of 21-bp repeats However, the relative con-tribution of these bZIP factors has not been compared directly in the same experimental setting Furthermore, it remains undetermined whether additional newly identi-fied bZIP proteins may also participate in Tax activation of 21-bp repeats

We next used dominant-negative proteins to assess the contributory roles of different bZIP transcription factors

on Tax-dependent activation We employed several well-documented dominant-negative inhibitors of CREB and Jun proteins including KCREB [32], A-CREB [33], A-Fos [34] and TAM67 [35] In addition, we constructed domi-nant-negative versions of ATF4 and LZIP [36] using the strategies suggested by Vinson et al [37] The dominant-inhibitory activities of the latter two proteins A-ATF4 and A-LZIP were verified using electrophoretic mobility shift assay and CAT reporter assay (data not shown) We interrogated these dominant negative bZIP proteins for inhibition of Tax activation of HTLV-I LTR (Fig 4A) All, KCREB, A-CREB, A-ATF4 and TAM67, suppressed Tax acti-vation in a dose-dependent manner (Fig 4A, lanes 3–10 compared to lane 2) However, different dominant nega-tive inhibitors constructed to the same protein using dif-ferent strategies might have difdif-ferent potencies For example, KCREB contains a mutation of a single amino acid in the CREB DNbinding domain [32], whereas A-CREB was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper region [33] Differential inhibitory effects

of KCREB and A-CREB were observed (Fig 4A, lanes 3–6)

In light of this, we quantitated and compared the inhibi-tory activities of dominant negative proteins all con-structed using the same strategy (Fig 4B) Since NFκB is not involved in Tax activation of HTLV-I LTR, we included

a dominant negative form of IKKβ, IKKβ DN, as a neutral control (Fig 4B, group 7) When we compared four dom-inant negative bZIP proteins, A-CREB, A-LZIP, A-Fos and A-ATF4, constructed using the identical molecular strat-egy, we observed the most dramatic suppression of Tax activation of HTLV-I LTR with A-CREB (Fig 4B, group 3, red column) The second most significant reduction in activity was seen with A-LZIP [36] (Fig 4B, group 6, red

Retrovirology 2004, 1:18 http://www.retrovirology.com/content/1/1/18

Relative responsiveness of enhancers to Tax in JPX9 cells

Figure 2

Relative responsiveness of enhancers to Tax in JPX9 cells (A) A representative example of CAT assay Tax-expressing plasmid

pIEX (1 µg) and increasing amounts (0.5 to 1 µg) of p21-HTLV-CAT (lanes 1 and 2), pCRE-HTLV-CAT (lanes 3 and 4), pAP1-HTLV-CAT (lanes 5 and 6), pSP1-pAP1-HTLV-CAT (lanes 7 and 8), pKB-pAP1-HTLV-CAT (lanes 9 and 10) and pSRE-pAP1-HTLV-CAT (lanes 11 and 12) were transfected into Jurkat cells CAT assays were performed 48 h after transfection AcCM: acetyl chloramphenicol

CM: chloramphenicol (B) Basal transcriptional activities of enhancer elements One microgram of plasmids containing the

HTLV TATAA alone (pHTLV-CAT; column 1) or the indicated enhancer elements (columns 2 to 7) were transfected into Jur-kat cells and the relative CAT activities were compared CAT activity from pKB-HTLV-CAT-transfected JurJur-kat cells was taken

as 100% (lane 6) (D) Tax-dependent transcriptional activities of enhancer elements The same plasmids as in C plus 1 µg of

Tax-expressing plasmid pIEX were co-transfected into Jurkat cells and the CAT assays were performed Fold activation in the presence of Tax versus in the absence of Tax was calculated and compared All CAT results are representative of three inde-pendent experiments

column) Thus, although several bZIP proteins can redundantly serve to mediate Tax-activation of the LTR, a clear preference for CREB is revealed by our assay

To verify the specificity of dominant negative effects, we also tested the activities of dominant negative proteins on

an NFκB-dependent reporter (Fig 4B, blue columns) Noticeably, none of the dominant negative bZIP proteins had an effect on Tax activation of NFκB (Fig 4B, groups 3–6 compared to group 2, blue columns) In contrast, the expression of IKKβ DN led to more than 50% suppression

of NFκB activity (Fig 4B, group 7, blue column) These results ruled out the possibility that CREB, ATF4, A-Fos and A-LZIP might non-specifically inhibit transcription

Functional significance of the HTLV-I TATAA element to transcriptional activation by Tax

In the course of our analyses, we noted that Tax can acti-vate the HTLV-I minimal TATAA-promoter without any known enhancer element by approximately 4-fold (Fig 1D, lane 1) This responsiveness of the HTLV-I minimal promoter is compatible with the concept that the core promoter can also be an important determinant of tran-scriptional specificity [2] We next asked whether all TATAA-elements are recognized by Tax in the same way for purposes of activated transcription Hence, we con-structed reporter plasmids that contain two 21-bp repeats and a minimal TATAA promoter from HTLV-I, HIV-1 or SV40 (Fig 5A) Since the TATAA promoters were all placed within the same context, we consider this a valid comparison of their relative responsiveness to Tax activation

While the basal activities of HIV-1 and SV40 minimal pro-moters were measurably greater than that from HTLV-I (Fig 5C), replacement of the HTLV-I TATAA with the counterpart element from either HIV-1 or SV40 led to a significant reduction in Tax responsiveness (Fig 5B, lanes 4–9; and Fig 5D) To further verify the importance of the TATAA-promoter, we asked the same question using a dif-ferent approach Above, Tax was recruited presumably to the downstream TATAA-box via factors bound to the HTLV-1 21bp repeats (see Fig 5A) We next investigated whether the same conclusion could be established if a Gal4 DNA-binding domain-Tax fusion protein (Gal4-Tax) was delivered to downstream TATAA element by tethering

to upstream Gal4-binding sites (see Fig 6A for reporter schematic) For this assay, we tested the HTLV-I, the

HIV-1, and the E1b TATAA-elements Consistent with the results from the 21 bp-TATAA experiments (Fig 5), Gal4-Tax activated most strongly the HTLV-I TATAA element (Fig 6B, lane 9 and Fig 6D, group 3) and was minimally potent for the adenoviral E1b promoter (Fig 6B, lane 7 and Fig 6D, group 1) As a control for Gal4-Tax, we

Differential activities of Tax mutants on 21-bp repeats (A),

κB (B), and SRE (C) motifs

Figure 3

Differential activities of Tax mutants on 21-bp repeats (A),

κB (B), and SRE (C) motifs One microgram of plasmid

expressing the indicated Tax mutants plus 5 µg of

p21-HTLV-CAT, pKB-HTLV-CAT or pSRE-HTLV-CAT was individually

transfected into HeLa cells CAT activity from wild type

Tax-transfected cells (lane 1) was taken as 100%

Retrovirology 2004, 1:18 http://www.retrovirology.com/content/1/1/18

checked in parallel the activity of the artificial Gal4-VP16

activator In contrast with Gal4-Tax, Gal4-VP16 showed

no preference for the various TATAA elements (Fig 6B,

lanes 4–6 and Fig 6D) Thus, two lines of evidence here

support that the HTLV-I TATAA promoter is an additional

Tax-specific responsive element

Evidence for Tax activity after assembly of an initiation

complex

Artificial recruitment of TBP to some higher eukaryotic

promoters bypasses transcriptional activation by a

DNA-tethered activator [21-24] When observed at such

pro-moters, this finding is evident that those activators act

mechanistically to enhance TBP recruitment to the TATAA

box For general transcriptional activation, additional

events subsequent to TBP recruitment are also known to

be functionally critical [21-23,25] To date, it remains

unclear whether Tax works transcriptionally through a

mechanism solely to recruit TBP or whether there are

additional mechanistic implications after TBP is recruited

to the TATAA-element

To investigate the mechanism(s) of Tax function with

respect to TBP recruitment, we constructed a series of

reporter plasmids (Fig 7A) with two copies of 21-bp

repeat, five copies of Gal4-binding sites and a minimal

TATAA sequence from one of four viral promoters

(HTLV-I, HIV-1, SV40 and E1b) We artificially delivered TBP to each promoter by provision of Gal4-TBP, and we asked whether Tax has an additional transcriptional effect which

is independent of TBP-recruitment to the TATAA-element

If Tax were to serve only for TBP-recruitment, then when TBP is tethered to the TATAA via Gal4-TBP one should expect to see no transcriptional enhancement from Tax Provocatively, for both the HTLV-I and HIV-1 TATAA ele-ments, Tax stimulated reporter expression greatly over that already achieved with Gal4-TBP (Fig 7, groups 1 and 2) Consistent with above findings, the SV40 and E1b TATAA elements appear to be transcriptionally rate-lim-ited by TBP recruitment, and Tax has minimal activity on these promoters However, the findings from the HTLV-I and the HIV-1 reporters provide evidence that more than simply accelerating TBP recruitment Tax can serve tran-scriptional function(s) subsequent to TBP (TFIID) assem-bly at the core promoter This is the first time that Tax has been shown to have a role subsequent to transcriptional initiation complex formation at the promoter

Discussion

Here, we have delineated functional requirements for both the TATAA promoter and the 21-bp enhancer ele-ments in HTLV-I Tax mediated activation of the viral LTR

Specific preference for CREB by Tax

Figure 4

Specific preference for CREB by Tax (A) An example of CAT assay HeLa cells were transfected with pU3RCAT alone (lane

1), pU3RCAT plus Tax expression plasmid pIEX (lane 2) or pU3RCAT plus pIEX plus increasing amounts (5 to 10 µg) of plas-mids expressing the indicated dominant-negative proteins (lanes 3–10) D-Threo-[dichloroacetyl-1-14C]-chloramphenicol was

as used as substrate in the CAT assay (B) Influence of dominant-negative proteins on Tax activation The cells received

pU3RCAT (red) or pKB-SV40-CAT (blue) only (group 1), pU3RCAT/pKB-SV40-CAT plus Tax-expressing plasmid pIEX (group 2) or pU3RCAT/pKB-SV40-CAT plus pIEX plus plasmids expressing the indicated dominant-negative proteins The empty vec-tor was used to normalize the amount of plasmids given to each group of cells DN: dominant-negative

Tax preferentially activates the HTLV-I minimal TATAA

promoter

Figure 5

Tax preferentially activates the HTLV-I minimal TATAA

pro-moter (A) CAT reporter plasmid Each plasmid contains

two 21-bp repeats and one copy of minimal promoter

(TATAA) from HTLV-I, HIV-1 and SV40 The minimal

pro-moter sequences are shown in blue (B) A representative

example of CAT assay The cells received 0, 0.5 and 1 µg of

Tax-expressing plasmid pIEX and 5 µg of the indicated CAT

reporter constructs (p21-HTLV-CAT, p21-HIV-CAT and

p21-SV40-CAT) (C, D) Basal and Tax-induced

transcrip-tional activities HeLa cells were co-transfected with 5 µg of

the indicated CAT reporter plasmids (HTLV-CAT,

p21-HIV-CAT and p21-SV40-CAT) plus 0.5 µg of pCMV empty

vector (w/o Tax) or pIEX (w/ Tax) Basal CAT activity from

p21-SV40-CAT-transfected cells was taken as 100% (C,

col-umn 3)

DNA-tethered Tax is specifically active on the HTLV-I mini-mal promoter

Figure 6

DNA-tethered Tax is specifically active on the HTLV-I

mini-mal promoter (A) CAT reporter plasmid Each plasmid

con-tains five tandem copies of Gal4-binding sites and one copy of minimal promoter (TATAA) from adenovirus E1b, HIV-1 and HTLV-I The minimal promoter sequences are shown in blue

(B) A representative example of CAT assay The cells were

co-transfected with 2 µg of a Gal4DB plasmid (pM vector alone for lanes 1–3, VP16 for lanes 4–6, and pGal4-Tax for lanes 7–9) and 5 µg of a CAT reporter construct (pG5-E1B-CAT for lanes 1, 4 and 7; pG5-HIV-CAT for lanes

2, 5 and 8; and pG5-HTLV-CAT for lanes 3, 6 and 9) (C, D)

Basal and activated transcriptional activities HeLa cells were co-transfected with 5 µg of the indicated CAT reporter plas-mids (pG5-E1B-CAT, pG5-HIV-CAT and pG5-HTLV-CAT)

plus 2 µg of pM empty vector (C), pGal4-VP16 (D, blue) or pGal4-Tax (D, yellow) Basal CAT activity from pG5-HIV-CAT-transfected cells was taken as 100% (C, column 2).

Retrovirology 2004, 1:18 http://www.retrovirology.com/content/1/1/18

To date Tax has been considered solely to initiate

tran-scription Our study shows for the first time that Tax has a

transcriptional role after assembly of an initiation

com-plex at the promoter

Preferential requirements for 21-bp repeats, CREB, and

the HTLV-I TATAA box

HTLV-I is etiologically associated with adult T-cell

leuke-mia [38,39] Expression of Tax leads to immortalization

of T lymphocytes [40-42] and transformation of rat

fibroblasts [43,44] Tax is a transcriptional activator that

can interact pleiotropically with several different

enhanc-ers In addition to the HTLV-I 21-bp repeats, κB and SRE

elements can also mediate Tax activation [4-6] Amongst

these three enhancers, it is clear that the viral 21-bp

repeats are the most highly responsive to Tax-activation

(Fig 1D) However, data elsewhere have raised questions

as to the identity of the 21-bp binding bZIP factor which

is best used to mediate Tax activation [30] In direct com-parisons, we have used matched A-CREB, A-Jun, A-ATF4 and A-LZIP dominant negative mutants to ask which bZIP factor is most contributory to Tax activation In our cell system, a novel bZIP factor called LZIP [36] can appar-ently participate in LTR transcription; however, for Tax activation CREB is preferred over ATF4 or c-Jun (Fig 4) Beyond the requirement for the 21-bp enhancer, our experiments revealed that the HTLV-I TATAA is also specif-ically preferred by Tax (Fig 5 and Fig 6) This finding is consistent with the general notion that core promoters can contribute specificity to transcriptional regulation [2] Indeed, core promoter preference by other cellular and viral activators such as Sp1, VP16 and Tat have been doc-umented previously [45-47] However, the reasons under-lying core promoter preferences are poorly understood TAFs have been suggested to be responsible for the core promoter selectivity of some activators [48-50] In this vein, the interaction of Tax with TBP [11] and TBP-associ-ated factors such as TAFII28 [51] might provide mechanis-tic explanations

Roles of Tax subsequent to TBP recruitment

A provocative notion which emerges from our study is that Tax can further activate a promoter at which TBP has already been artificially tethered (Fig 7) Experiments in yeast and mammalian cells indicate that many genes can

be activated through artificial recruitment of TBP and other components of the basal transcription machinery to their promoters [52,53] In yeast, artificial recruitment of TBP bypasses the effect of DNA-tethered activators whereas the activators fail to activate transcription when physically fused to components of the basal transcription machinery [54] This and other lines of evidence support the notion that activator-dependent recruitment of TBP and basal transcription machinery is a major mechanism for transcriptional activation in yeast cells [54,55] In con-trast, artificial recruitment of TBP to mammalian promoters has not yet been extensively studied Among the few promoters examined, some such as the ones from E1b and thymidine kinase genes can be fully activated by artificially recruited TBP, while others such as HIV-1 and c-fos promoters are stimulated weakly [21-25] On the other hand, some activators such as VP16, E1A, Tat, E2F1 and IE2 work synergistically with artificially recruited TBP, while others such as Sp1 cannot further enhance the activ-ity of DNA-tethered TBP [21,22] Thus, artificial recruit-ment of TBP might insufficiently activate transcription in mammalian cells and different activators might function

at different steps with respect to TBP recruitment Our results indicate that DNA-bound TBP can activate HTLV-I LTR only weakly, but its activity is further enhanced by Tax (Fig 6) While such experimental results do not exclude that under physiological circumstances the primary

Tax further activates a promoter with DNA-tethered TBP

Figure 7

Tax further activates a promoter with DNA-tethered TBP

(A) CAT reporter plasmid Each plasmid contains two copies

of 21-bp repeat, five copies of Gal4-binding sites and one

copy of minimal promoter (TATAA) from adenovirus

HTLV-I, HIV-1, SV40 and adenovirus E1b (B) CAT assay HeLa cells

were co-transfected with 5 µg of the indicated CAT reporter

plasmids (HTLV-CAT, HIV-CAT,

p21-G5-SV40-CAT and p21-G5-E1B-CAT) and 2 µg of pGal4-TBP

(yellow) or 2 µg of pIEX (Tax; pink) or 2 µg of pGal4-TBP

plus 2 µg of pIEX (Gal4-TBP + Tax; blue) Basal CAT activity

from cells transfected with pGal4-TBP plus

p21-G5-E1B-CAT was taken as 100% (group 4, yellow)

function of Tax may be to enhance initiation complex

formation (i.e TBP-recruitment), they do indicate that

Tax has an additional transcriptional activity that extends

to phases after transcriptional initiation Currently, we do

not know whether this is at the step of promoter

clear-ance, transcriptional elongation, or some other processes

However, we do believe that Tax should be added to the

list of mammalian activators that can function at steps

subsequent to TBP recruitment [21-25]

All the transcriptional assays in the present study were

based on transiently transfected reporters We noted that

transiently transfected and stably integrated promoters

might behave differently [24,56] Obviously, chromatin

structure and copy numbers can account for significant

differences [56,57] Future experiments are required to

verify whether the observations established here also hold

for stably integrated HTLV-I LTRs

Methods

Plasmids

Chloramphenicol acetyltransferase (CAT) reporter

plas-mid pG5CAT was from Clontech CAT plasplas-mid pU3RCAT

containing the HTLV-I LTR has been previously described

[13] Other CAT plasmids were derived from pCAT-basic

(Promega) For each construct, one copy of a minimal

promoter and two copies of an enhancer were chemically

synthesized and cloned into pCAT-basic For example,

pCRE-HTLV-CAT contains two copies of canonical CRE

motif plus one copy of HTLV-I minimal promoter (Fig

1A) Five copies of Gal4-binding sites as in pG5CAT were

also inserted in some reporters All constructs have the

same spacing between the TATAA box and the CAT open

reading frame (44 bp) or between the enhancer and the

TATAA box (23 bp)

Sequences of canonical CRE, Sp1, AP1 and κB motifs in

the reporter plasmids have been described [36,58,59]

HTLV-I 21-bp repeats and serum response element (SRE)

in the plasmids were derived from the following synthetic

oligonucleotides: 21-bp repeats, 5'-AGCTTAGGCC

CTGACGTGTCCCCCTGGATCCTAGGCCCTGACGTGTC-CCCCTA-3' and 5'-AGCTTAG

GGGGACACGTCAG-GGCCTAGGATCCAGGGGGACACGTCAGGGCCTA-3';

SRE, 5'-AGCTACCATATTAGGATCCATATTAGGT-3' and

5'-AGCTACCTAATATGGATCCTAATATGGT-3' Sequences

of the minimal promoter elements from HTLV-I, HIV-1,

SV40 and adenoviral E1b have been described [60] The

SV40 early promoter naturally used for expression of the

viral T/t antigens was used

Expression plasmids for wild type and mutant Tax have

been described elsewhere [26,61] pIEX is a Tax expression

vector driven by cytomegalovirus IE promoter Tax

mutants are indicated by the amino acid to be changed,

the position of the residue, and the replacement amino acid (e.g Tax S150A) Amino acids that were removed in mutants are indicated as in Tax ∆3–6 Expression vector

pM for Gal4 DNA binding domain (Gal4DB; amino acids 1–147) was from Clontech Tax, human TBP and the acti-vation domain of VP16 fused to Gal4DB were designated Gal4-Tax, Gal4-TBP and Gal4-VP16, respectively Expres-sion plasmids for Gal4-Tax and Gal4-TBP have been described [10,21] Expression plasmid for Gal4-VP16 was from Clontech

Expression plasmid pRSV-KCREB for the dominant-nega-tive CREB protein KCREB [32] was kindly provided by Dr Richard Goodman Expression plasmids pCMV-ACREB and pCMV-AFOS for dominant-negative CREB and AP1 proteins A-CREB [33] and A-Fos [34] were gifts from Dr Charles Vinson Expression plasmid pCMV-TAM67 for dominant-negative c-Jun protein TAM67 [35] was from

Dr Michael Birrer Expression plasmids pCMV-AATF4 and pCMV-ALZIP for dominant-negative ATF4 and LZIP proteins A-ATF4 and A-LZIP were derived from pCMV500 provided by Dr Charles Vinson [33,37] A-ATF4 contains 304–352 amino acids of human ATF4 and A-LZIP con-tains 175–223 amino acids of human LZIP A-ATF4 and A-LZIP can specifically and dominantly inhibit the CRE-binding and CRE-activating activities of ATF4 and LZIP, respectively, in electrophoretic mobility shift assay and CAT reporter assay (data not shown) Expression plasmid for dominant-negative IKKβ (IKKβ DN) was a gift from

Dr Michael Karin [62]

Reporter assay

HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with fetal calf serum and antibiot-ics, seeded at 5 × 105 cells/well into six-well culture plates and transfected using calcium phosphate method as described [13] Jurkat cells were cultured in RPMI 1640 medium and transfected by FUGENE 6 reagents (Roche) CAT activity was assayed as previously described [63] Briefly, transfected cells were harvested and lysed by freez-ing and thawfreez-ing Protein concentration of clarified lysates was determined by Bradford reagent (Bio-Rad) Equal amounts of lysates were mixed with 14C-labeled chloram-phenicol (Amersham) and acetyl coenzyme A (Calbio-chem) for CAT reaction CAT activities were detected using thin-layer chromatography and quantified by phosphorimager (Molecular Dynamics) For transfection

of cells, each well received the same dose of plasmids The empty vector or pUC19 was added to compensate for the different amounts of plasmids when necessary

Competing interests

None declared